Team:KIT-Kyoto/c6h12o6

From 2012.igem.org

(Difference between revisions)
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<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td></Td><Td>sample1</Td></Tr>
+
<Tr><Td>　　</Td><Td>　sample1　</Td></Tr>
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<Tr><Td>10ng/μL TNFAIP3</Td><Td>6μL</Td></Tr>
+
<Tr><Td>　10ng/μL TNFAIP3　</Td><Td>　6μL　</Td></Tr>
-
<Tr><Td>10× KOD plus buffer</Td><Td>10μL</Td></Tr>
+
<Tr><Td>　10× KOD plus buffer　</Td><Td>　10μL　</Td></Tr>
-
<Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr>
+
<Tr><Td>　2mM dNTPs　</Td><Td>　10μL　</Td></Tr>
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>3.2μL</Td></Tr>
+
<Tr><Td>　25mM MgSO<sub>4</sub>　</Td><Td>　3.2μL　</Td></Tr>
-
<Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr>
+
<Tr><Td>　10P 5'primer　</Td><Td>　3μL　</Td></Tr>
-
<Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr>
+
<Tr><Td>　10P 3'primer　</Td><Td>　3μL　</Td></Tr>
-
<Tr><Td>KOD plus</Td><Td>2μL</Td></Tr>
+
<Tr><Td>　KOD plus　</Td><Td>　2μL　</Td></Tr>
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<Tr><Td>dH<sub>2</sub>O</Td><Td>62.8μL</Td></Tr>
+
<Tr><Td>　dH<sub>2</sub>O　</Td><Td>　62.8μL　</Td></Tr>
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<Tr><Td>total</Td><Td>100μL</Td></Tr>
+
<Tr><Td>　total　</Td><Td>　100μL　</Td></Tr>
</Table>
</Table>
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<Tr><Td>　temperature　</Td><Td>　time　</Td><Td>　cycle　</Td></Tr>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<Tr><Td>　95°C　</Td><Td>　2min　</Td><Td>　　</Td></Tr>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td>　95°C　</Td><Td>　15sec　</Td><Td>　25cycle　</Td></Tr>
-
<Tr><Td>60°C</Td><Td>30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td>　60°C　</Td><Td>　30sec　</Td><Td>　25cycle　</Td></Tr>
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td>　68°C　</Td><Td>　2min30sec　</Td><Td>　25cycle　</Td></Tr>
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td></Td></Tr>
+
<Tr><Td>　68°C　</Td><Td>　2min30sec　</Td><Td>　　</Td></Tr>
-
<Tr><Td>14°C</Td><Td>∞</Td><Td></Td></Tr>
+
<Tr><Td>　14°C　</Td><Td>　∞　</Td><Td>　　</Td></Tr>
</Table>
</Table>
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td></Td><Td>sample1</Td><Td>sample2</Td><Td>sample3</Td><Td>sample4</Td></Tr>
+
<Tr><Td>　　</Td><Td>　sample1　</Td><Td>　sample2　</Td><Td>　sample3　</Td><Td>　sample4　</Td></Tr>
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<Tr><Td>10ng/μL API2-MALT1</Td><Td>2μL</Td><Td>2μL</Td><Td>1μL</Td><Td>1μL</Td></Tr>
+
<Tr><Td>　10ng/μL API2-MALT1　</Td><Td>　2μL　</Td><Td>　2μL　</Td><Td>　1μL　</Td><Td>　1μL　</Td></Tr>
-
<Tr><Td>10× KOD plus buffer</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td></Tr>
+
<Tr><Td>　10× KOD plus buffer　</Td><Td>　2μL　</Td><Td>　2μL　</Td><Td>　2μL　</Td><Td>　2μL　</Td></Tr>
-
<Tr><Td>2mM dNTPs</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td></Tr>
+
<Tr><Td>　2mM dNTPs　</Td><Td>　2μL　</Td><Td>　2μL　</Td><Td>　2μL　</Td><Td>　2μL　</Td></Tr>
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>0.8μL</Td><Td>0.8μL</Td><Td>0.8μL</Td><Td>0.8μL</Td></Tr>
+
<Tr><Td>　25mM MgSO<sub>4</sub>　</Td><Td>　0.8μL　</Td><Td>　0.8μL　</Td><Td>　0.8μL　</Td><Td>　0.8μL　</Td></Tr>
-
<Tr><Td>10P 5'primer</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td></Tr>
+
<Tr><Td>　10P 5'primer　</Td><Td>　0.6μL　</Td><Td>　0.6μL　</Td><Td>　0.6μL　</Td><Td>　0.6μL　</Td></Tr>
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<Tr><Td>10P 3'primer</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td></Tr>
+
<Tr><Td>　10P 3'primer　</Td><Td>　0.6μL　</Td><Td>　0.6μL　</Td><Td>　0.6μL　</Td><Td>　0.6μL　</Td></Tr>
-
<Tr><Td>KOD plus</Td><Td>0.4μL</Td><Td>0.4μL</Td><Td>0.4μL</Td><Td>0.4μL</Td></Tr>
+
<Tr><Td>　KOD plus　</Td><Td>　0.4μL　</Td><Td>　0.4μL　</Td><Td>　0.4μL　</Td><Td>　0.4μL　</Td></Tr>
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<Tr><Td>dH<sub>2</sub>O</Td><Td>11.6μL</Td><Td>11.6μL</Td><Td>12.6μL</Td><Td>12.6μL</Td></Tr>
+
<Tr><Td>　dH<sub>2</sub>O　</Td><Td>　11.6μL　</Td><Td>　11.6μL　</Td><Td>　12.6μL　</Td><Td>　12.6μL　</Td></Tr>
-
<Tr><Td>total</Td><Td>20μL</Td><Td>20μL</Td><Td>20μL</Td><Td>20μL</Td></Tr>
+
<Tr><Td>　total　</Td><Td>　20μL　</Td><Td>　20μL　</Td><Td>　20μL　</Td><Td>　20μL　</Td></Tr>
</Table>
</Table>
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<Tr><Td>　temperature　</Td><Td>　time　</Td><Td>　cycle　</Td></Tr>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<Tr><Td>　95°C　</Td><Td>　2min　</Td><Td>　　</Td></Tr>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td>　95°C　</Td><Td>　15sec　</Td><Td>　25cycle　</Td></Tr>
-
<Tr><Td>50°C(sample1,3)or55°C(sample2,4)</Td><Td>30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td>　50°C(sample1,3)or55°C(sample2,4)　</Td><Td>　30sec　</Td><Td>　25cycle　</Td></Tr>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td>　68°C　</Td><Td>　3min30sec　</Td><Td>　25cycle　</Td></Tr>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td></Td></Tr>
+
<Tr><Td>　68°C　</Td><Td>　3min30sec　</Td><Td>　　</Td></Tr>
-
<Tr><Td>14°C</Td><Td>∞</Td><Td></Td></Tr>
+
<Tr><Td>　14°C　</Td><Td>　∞　</Td><Td>　　</Td></Tr>
</Table>
</Table>
<br>
<br>

Revision as of 00:00, 25 September 2012

August 1st


*TNFA and API2

　　　sample1　
　10ng/μL TNFAIP3　　6μL　
　10× KOD plus buffer　　10μL　
　2mM dNTPs　　10μL　
　25mM MgSO4　　3.2μL　
　10P 5'primer　　3μL　
　10P 3'primer　　3μL　
　KOD plus　　2μL　
　dH2O　　62.8μL　
　total　　100μL　

　temperature　　time　　cycle　
　95°C　　2min　　　
　95°C　　15sec　　25cycle　
　60°C　　30sec　　25cycle　
　68°C　　2min30sec　　25cycle　
　68°C　　2min30sec　　　
　14°C　　∞　　　

　　　sample1　　sample2　　sample3　　sample4　
　10ng/μL API2-MALT1　　2μL　　2μL　　1μL　　1μL　
　10× KOD plus buffer　　2μL　　2μL　　2μL　　2μL　
　2mM dNTPs　　2μL　　2μL　　2μL　　2μL　
　25mM MgSO4　　0.8μL　　0.8μL　　0.8μL　　0.8μL　
　10P 5'primer　　0.6μL　　0.6μL　　0.6μL　　0.6μL　
　10P 3'primer　　0.6μL　　0.6μL　　0.6μL　　0.6μL　
　KOD plus　　0.4μL　　0.4μL　　0.4μL　　0.4μL　
　dH2O　　11.6μL　　11.6μL　　12.6μL　　12.6μL　
　total　　20μL　　20μL　　20μL　　20μL　

　temperature　　time　　cycle　
　95°C　　2min　　　
　95°C　　15sec　　25cycle　
　50°C(sample1,3)or55°C(sample2,4)　　30sec　　25cycle　
　68°C　　3min30sec　　25cycle　
　68°C　　3min30sec　　　
　14°C　　∞　　　

August 2nd


*TNFA and API2

1-21-3
DNA sample10μL20μL
6×Dye2μL4μL
total12μL25μL




1-2
DNA sample90μL
6×Dye18μL
total108μL






10ng/μL API2-MALT15μL
10×KOD plus buffer10μL
2mM dNTPs10μL
25mM MgSO44μL
10P 5'primer3μL
10P 3'primer3μL
KOD plus2μL
dH2O63μL
total100μL

temperaturetimecycle
95°C2min
95°C15sec35cycle
55°C30sec35cycle
68°C3min30sec35cycle
68°C3min30sec
14°C∞

Result



August 3rd


*TNFA and API2

attB TNFAIP3(80ng/μL)1μL
pDONR(455ng/μL)0.35μL
TE buffer7.65
total9μL

sample1sample2
10ng/μL AIP2-MALT12.5μL2.5μL
10×KOD plus buffer5μL5μL
2mM dNTPs5μL5μL
25mM MgSO42μL2.4μL
10P 5'primer1.5μL1.5μL
10P 3'primer1.5μL1.5μL
KOD plus1μL1μL
dH231.5μL31.1μL
total50μL50μL

temperaturetimecycle
95°C2min
95°C15sec35cycle
55°C30sec35cycle
68°C3min30sec35cycle
68°C3min30sec
14°C∞




August 4th


*TNFA and API2

August 5th


*TNFA and API2





August 6th


*TNFA and API2

August 7th


*TNFA and API2





pDONRBP TNFA-1BP TNFA-2
DNA sample1μL1μL1μL
6×Dye1μL1μL1μL
dH2O4μL4μL4μL
total6μL6μL6μL

BP TNFA-22μL
pTFW(Destination vector)0.5μL
TE buffer6.5μL
total9μL

WEEK１終わり

August 8th


*TNFA and API2

August 9th


*TNFA and API2

DNA sample1μL
10×H buffer0.5μL
EcoRⅠ0.2μL
dH2O3.3μL
total5μL




1-2
50ng/μL LR TNFA-11μL
10×rTaq buffer2μL
2mM dNTPs2μL
25mM MgCl20.8μL
10P 5'primer0.6μL
10P 3'primer0.6μL
rTaq0.4μL
dH2O12.6μL
total20μL

temperaturetimecycle
95°C2min
95°C15sec25cycle
55°C30sec25cycle
68°C2min30sec25cycle
68°C2min30sec
14°C∞

August 10th


*TNFA and API2





August 13th


*TNFA and API2

1-11μL
10×M buffer0.5μL
Hind Ⅲ0.2μL
dH2O3.3μL
total5μL




August 14th


*TNFA and API2

1-1sample1
10ng/μL TNFAIP36μL
10×KOD plus buffer10μL
2mM dNTPs10μL
25mM MgSO43.2μL
10P 5'primer3μL
10P 3'primer3μL
KOD plus2μL
dH2O62.8μL
total100μL

temperaturetimecycle
95°C2min
95°C15sec25cycle
60°C30sec25cycle
68°C2min30sec25cycle
68°C2min30sec
14°C∞




WEEK2終わり

August 20th


TNFA and API2

1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition
a product of PCR attB TNFAIP3(8/1)
DNA sample90μL
6×Dye18μL
Total108μL

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

Results
We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)

August 21st


TNFA and API2

1. Density measurement of attB TNFAIP3
We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )

Results We estimated attB TNFAIP3(we make this time) is 35ng/uL

2. BP reaction
We adjusted solution (vials) on 1.5mL tube to next composition.
attB TNFAIP3(35ng/μL)2μL
pDONR(150ng/μL)1μL
TE buffer5μL
Total8μL

We added BP Clonase Ⅱ enzyme mix-2uL to this vials.
We incubated these vials for 2hour at 25˚C. Next we did BP reaction

3. Transformation
We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.
Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(＋) and we keep 37℃ overnight.

August 22nd


TNFA and API2

We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .
We cultivated these colonies on 2.5mL Kanamycin(＋) LB culture medium and we kept shaking this medium overnight.

Retrieved from "http://2012.igem.org/Team:KIT-Kyoto/c6h12o6"